背景:确定药物结合靶标及其相应位点对于药物发现和机制研究至关重要。有限的蛋白水解耦合质谱(LiP-MS)是一种用于检测化合物和蛋白质相互作用的复杂方法。然而,在某些情况下,由于结构变化小或缺乏低丰度蛋白的富集,LiP-MS无法识别靶蛋白。为了克服这个缺点,我们开发了一种热稳定性辅助的有限蛋白水解耦合质谱(TALiP-MS)方法,用于有效的药物靶标发现.
结果:我们证明了新策略,TALiP-MS,可以有效地识别各种配体的靶蛋白,包括环孢菌素A(钙调磷酸酶抑制剂),格尔德霉素(HSP90抑制剂),和星形孢菌素(激酶抑制剂),准确识别药物结合域。TALiP方案使LiP-MS实验中检测到的靶肽的数量增加2至8倍。同时,TALiP-MS方法不仅可以鉴定配体结合稳定性和不稳定蛋白,而且与热蛋白质组分析(TPP)和基于机器学习的有限蛋白水解(LiP-Quant)方法具有高度互补性。开发的TALiP-MS方法用于鉴定雷公藤红素(CEL)的靶蛋白,一种天然产品,以其强大的抗氧化和抗癌血管生成作用而闻名。其中,四种蛋白质,MTHFD1,UBA1,ACLY,通过使用细胞热转移测定进一步验证了SND1对CEL的强亲和力。此外,CEL诱导的不稳定蛋白如TAGLN2和CFL1也得到了验证。
结论:总的来说,这些发现强调了TALiP-MS方法识别药物靶标的有效性,阐明结合位点,甚至检测药物诱导的复杂蛋白质组中靶蛋白质的构象变化。
BACKGROUND: Identifying drug-binding targets and their corresponding sites is crucial for drug discovery and mechanism studies. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a sophisticated method used for the detection of compound and protein interactions. However, in some cases, LiP-MS cannot identify the target proteins due to the small structure changes or the lack of enrichment of low-abundant protein. To overcome this drawback, we developed a thermostability-assisted limited proteolysis-coupled mass spectrometry (TALiP-MS) approach for efficient drug target discovery.
RESULTS: We proved that the novel strategy, TALiP-MS, could efficiently identify target proteins of various ligands, including cyclosporin A (a calcineurin inhibitor), geldanamycin (an HSP90 inhibitor), and
staurosporine (a kinase inhibitor), with accurately recognizing drug-binding domains. The TALiP protocol increased the number of target peptides detected in LiP-MS experiments by 2- to 8-fold. Meanwhile, the TALiP-MS approach can not only identify both ligand-binding stability and destabilization proteins but also shows high complementarity with the thermal proteome profiling (TPP) and machine learning-based limited proteolysis (LiP-Quant) methods. The developed TALiP-MS approach was applied to identify the target proteins of celastrol (CEL), a natural product known for its strong antioxidant and anti-cancer angiogenesis effect. Among them, four proteins, MTHFD1, UBA1, ACLY, and SND1 were further validated for their strong affinity to CEL by using cellular thermal shift assay. Additionally, the destabilized proteins induced by CEL such as TAGLN2 and CFL1 were also validated.
CONCLUSIONS: Collectively, these findings underscore the efficacy of the TALiP-MS method for identifying drug targets, elucidating binding sites, and even detecting drug-induced conformational changes in target proteins in complex proteomes.